Broad Kinase Inhibition Mitigates Early Neuronal Dysfunction in Tauopathy.

Tauopathies are a group of more than twenty known disorders that involve progressive neurodegeneration, cognitive decline and pathological tau accumulation. Current therapeutic strategies provide only limited, late-stage symptomatic treatment. This is partly due to lack of understanding of the molecular mechanisms linking tau and cellular dysfunction, especially during the early stages of disease progression. In this study, we treated early stage tau transgenic mice with a multi-target kinase inhibitor to identify novel substrates that contribute to cognitive impairment and exhibit therapeutic potential. Drug treatment significantly ameliorated brain atrophy and cognitive function as determined by behavioral testing and a sensitive imaging technique called manganese-enhanced magnetic resonance imaging (MEMRI) with quantitative R1 mapping. Surprisingly, these benefits occurred despite unchanged hyperphosphorylated tau levels. To elucidate the mechanism behind these improved cognitive outcomes, we performed quantitative proteomics to determine the altered protein network during this early stage in tauopathy and compare this model with the human Alzheimer's disease (AD) proteome. We identified a cluster of preserved pathways shared with human tauopathy with striking potential for broad multi-target kinase intervention. We further report high confidence candidate proteins as novel therapeutically relevant targets for the treatment of tauopathy. Proteomics data are available via ProteomeXchange with identifier PXD023562.

PERK inhibition delays neurodegeneration and improves motor function in a mouse model of Marinesco-Sjögren syndrome.

Marinesco-Sjögren syndrome (MSS) is a rare, early onset, autosomal recessive multisystem disorder characterized by cerebellar ataxia, cataracts and myopathy. Most MSS cases are caused by loss-of-function mutations in the gene encoding SIL1, a nucleotide exchange factor for the molecular chaperone BiP which is essential for correct protein folding in the endoplasmic reticulum. Woozy mice carrying a spontaneous Sil1 mutation recapitulate key pathological features of MSS, including cerebellar atrophy with degeneration of Purkinje cells and progressive myopathy. Because the PERK branch of the unfolded protein response is activated in degenerating neurons of woozy mice, and inhibiting PERK-mediated translational attenuation has shown protective effects in protein-misfolding neurodegenerative disease models, we tested the therapeutic efficacy of GSK2606414, a potent inhibitor of PERK. Mice were chronically treated with GSK2606414 starting from a presymptomatic stage, and the effects were evaluated on biochemical, histopathological and clinical readouts. GSK2606414 delayed Purkinje cell degeneration and the onset of motor deficits, prolonging the asymptomatic phase of the disease; it also reduced the skeletal muscle abnormalities and improved motor performance during the symptomatic phase. The protein but not the mRNA level of ORP150, a nucleotide exchange factor which can substitute for SIL1, was increased in the cerebellum of GSK2606414-treated woozy mice, suggesting that translational recovery promoted the synthesis of this alternative BiP co-factor. Targeting PERK signaling may have beneficial disease-modifying effects in carriers of SIL1 mutations.

Effects of Rho-kinase inhibition on myosin light chain phosphorylation and obstruction-induced detrusor overactivity.

OBJECTIVES: To study the relationship between myosin light chain phosphorylation of the detrusor muscle and spontaneous smooth muscle contractions in a rabbit model of partial outlet obstruction. METHODS: New Zealand white rabbit urinary bladders were partially obstructed for 2 weeks. Rabbits were euthanized, detrusor muscle strips were hung on a force transducer and spontaneous activity was measured at varying concentrations (0-0.03 µM/L) of the Rho-kinase inhibitors GSK 576371 or 0.01 µM/L Y27632. Basal myosin light chain phosphorylation was measured by 2-D gel electrophoresis in control and GSK 576371-treated strips. RESULTS: Both drugs suppressed the force of spontaneous contractions, whereas GSK 576371 had a more profound effect on the frequency of the contractions. The IC50 values for the inhibition of frequency and force of spontaneous contractions were 0.17 µM/L and 0.023 µM/L for GSK 576371, respectively. The compound significantly decreased the basal myosin light chain phosphorylation from 28.0 ± 3.9% to 13.5 ± 1.9% (P < 0.05). At 0.01 µM/L, GSK 576371 inhibited spontaneous bladder overactivity by 50%, but inhibited carbachol-elicited contractions force by just 25%. CONCLUSIONS: These data suggest that Rho-kinase regulation of myosin light chain phosphorylation contributes to the spontaneous detrusor activity induced by obstruction. This finding could have therapeutic implications by providing another therapeutic option for myogenic, overactive bladder.

Improving the developability profile of pyrrolidine progesterone receptor partial agonists.

The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein.

Urodynamic measurements by radiotelemetry in conscious, freely moving beagle dogs.

PURPOSE: Urodynamics have been traditionally recorded in anesthetized or conscious animals implanted with a bladder catheter that is used to artificially fill the bladder while measuring intravesicular bladder pressure. Anesthesia alters the urodynamics and in the conscious state this methodology requires that the dogs be tethered/restrained, which evokes stress and limits the period of continuous urodynamic assessment. A more physiological and chronic method of evaluating pharmacological responses on urodynamics is necessary. MATERIALS AND METHODS: Adult female beagle dogs were surgically instrumented with radiotelemetry transmitters enabling urodynamic/hemodynamic recordings. Telemetered urodynamics were compared to those measured in anesthetized dogs receiving bladder infusion of saline. The response to diuresis with furosemide (Intervet, Millsboro, Delaware) and the M3 selective antimuscarinic darifenacin (Matrix Laboratories, Hyderabad, India) were evaluated. RESULTS: Saline infused, anesthetized dogs demonstrated lower peak micturition pressure and higher threshold pressure than conscious, freely moving telemetered dogs. In telemetered dogs a single dose of furosemide increased voiding frequency and average urine volume per void. Darifenacin decreased peak voiding pressure without affecting voiding frequency. CONCLUSION: Telemetry provides the potential to significantly decrease animal use while enabling the continuous monitoring of urodynamics under more physiological conditions without tethering or artificial filling. In addition, this new model facilitates evaluation of the chronic efficacy of new urological therapies.

Synthesis and SAR of amino acid-derived heterocyclic progesterone receptor full and partial agonists.

Two classes of amino acid-derived heterocyclic progesterone receptor ligands were developed to address the metabolic issues posed by the dimethyl amide functionality of the lead compound (1). The tetrazole-derived ligands behaved as potent partial agonists, while the 1,2,4-triazole ligands behaved as potent full agonists.

Rational design of orally-active, pyrrolidine-based progesterone receptor partial agonists.

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors.

The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.

Evaluation of pressor and visceromotor reflex responses to bladder distension in urethane anesthetized rats.

AIMS: We tested cardiovascular and visceromotor reflex (VMR) responses to urinary bladder distension (UBD) in urethane anesthetized rats to see if it can replicate the response pattern and the inhibition of bladder nociceptive transmission by analgesics seen in isoflurane anesthetized animals. METHODS: Female Sprague-Dawley rats under 3% isoflurane anesthesia were acutely instrumented with jugular venous, carotid arterial, and bladder cannulas for drug administration, blood pressure (BP) measurement, and bladder distension, respectively. Needle electrodes were placed directly into the abdominal musculature to measure myoelectrical activity subsequent to phasic UBD (30 sec in 3 min intervals). A cardiovascular response (pressor) and a VMR response (a contraction of abdominal and hind limb musculature) to UBD were evaluated in urethane (1.2 g/kg, i.v.) or isoflurane (1%) anesthetized rats. RESULTS: Pressor and VMR responses to noxious UBD (60 mmHg) were generated under both anesthesics. The thresholds of stimulus response functions for both pressor and VMR responses were not affected by either anesthesics. However, the magnitude of the maximal pressor response was significantly reduced in urethane anesthesia. The analgesics, morphine, and mexiletine, significantly inhibited the VMR response to noxious UBD under both anesthetics, but the intensities of the inhibition from both analgesics under urethane anesthesia were much lower than under isoflurane anesthesia (ID50: 2.07 mg/kg vs. 0.88 mg/kg for morphine, >10 mg/kg vs. 0.47 mg/kg for mexiletine). CONCLUSIONS: The rat urinary bladder distension model in urethane anesthetized rats demonstrates a blunted maximal pressor response and a reduced inhibition of visceral nociceptive transmission by analgesics. Neurourol. Urodynam. 28:442-446, 2009. (c) 2008 Wiley-Liss, Inc.

Sustained elevation of MG53 in the bloodstream increases tissue regenerative capacity without compromising metabolic function.

MG53 is a muscle-specific TRIM-family protein that presides over the cell membrane repair response. Here, we show that MG53 present in blood circulation acts as a myokine to facilitate tissue injury-repair and regeneration. Transgenic mice with sustained elevation of MG53 in the bloodstream (tPA-MG53) have a healthier and longer life-span when compared with littermate wild type mice. The tPA-MG53 mice show normal glucose handling and insulin signaling in skeletal muscle, and sustained elevation of MG53 in the bloodstream does not have a deleterious impact on db/db mice. More importantly, the tPA-MG53 mice display remarkable dermal wound healing capacity, enhanced muscle performance, and improved injury-repair and regeneration. Recombinant human MG53 protein protects against eccentric contraction-induced acute and chronic muscle injury in mice. Our findings highlight the myokine function of MG53 in tissue protection and present MG53 as an attractive biological reagent for regenerative medicine without interference with glucose handling in the body.

Effects of the beta 3-adrenergic receptor agonist disodium 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316243) on bladder micturition reflex in spontaneously hypertensive rats.

The present study investigated whether beta3-adrenoceptor activation acts on the bladder afferent pathway by examination of the visceromotor reflex (VMR) and pressor responses to urinary bladder distension (UBD) and whether beta3-adrenoceptor activation produces urinary bladder relaxation in hyperactive spontaneously hypertensive rats (SHRs) in comparison with their normotensive control rats [Wistar-Kyoto (WKY)]. Using the VMR responses to noxious UBD as a measure of bladder afferent signal transmission, SHRs did not present a sensitized bladder phenotype. However, reduced bladder compliance accompanied by a reduced void threshold was detected in the SHR detrusor. Furthermore, the selective beta3-adrenoceptor agonist disodium 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316243) (i.v.) failed to attenuate VMR or pressor responses to UBD in either SHRs or WKY rats, but it dose-dependently inhibited rhythmic contraction (RC) in SHRs. The minimal effective dose was 0.001 mg/kg. Using the same model in WKY rats, CL-316243 did not elicit significant inhibition of contractions in the bladder RC assay. These results suggest that SHRs represent abnormal efferent/detrusor function (detrusor overactivity) without mechanosensory afferent hypersensitivity. The beta3-adrenoceptor agonist CL-316243 acts on the detrusor muscle to increase urine storage in SHRs.

N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I.

The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4+/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4-/- bladders. TRPV4 activation with GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4-/- bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4alpha-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4+/+ and TRPV4-/- mice revealed an enhanced bladder capacity in the TRPV4-/- mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in TRPV4-/- mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM.

Role of spinal Cav2.2 and Cav2.1 ion channels in bladder nociception.

PURPOSE: High voltage activated calcium channels have been implicated in nociceptive transmission in several animal pain models. To our knowledge this is the first study to evaluate the ability of various high voltage activated calcium channel blockers to inhibit the transmission of noxious stimuli from the bladder at the level of the spinal cord. MATERIALS AND METHODS: The nociceptive response was measured by analyzing the visceromotor reflex and cardiovascular (pressor) responses to bladder distention. The role of Cav2.2 (N-type), Cav2.1 (P/Q-type) and Cav1 (L-type) calcium channels in bladder nociceptive reflex responses was examined using omega-conotoxin-GVIA, omega-agatoxin IVA/omega-conotoxin MVIIC and verapamil (Sigma-Aldrich), respectively. Female Sprague-Dawley rats were acutely instrumented with intrathecal catheters, carotid arterial and bladder cannulas. Needle electrodes were placed directly into the abdominal musculature to measure myoelectric activity subsequent to repeat phasic bladder distention at 60 mm Hg for 30 seconds at 3-minute intervals with the rats under 1% isoflurane. Drugs were administered by intrathecal injection 2 minutes before distention and responses were recorded for 15 minutes per dose. RESULTS: When administered intrathecally, omega-conotoxin-GVIA and omega-conotoxin MVIIC (10 microg/kg each) significantly attenuated reflex responses to noxious bladder distention to 12% and 65% of the maximal visceromotor reflex response, and to 45% and 59% of the control pressor response, respectively. However, agatoxin and verapamil were less effective. CONCLUSIONS: The study suggests that spinal Cav2.2 and Q-type Cav2.1 calcium channels contribute to acute bladder nociception, while Cav1 channels have a limited role.

Tumor-specific efficacy of transforming growth factor-beta RI inhibition in Eker rats.

PURPOSE: Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions. EXPERIMENTAL DESIGN: We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency. RESULTS: Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals. CONCLUSION: Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator.

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.

TLR2 agonism reverses chemotherapy-induced neutropenia in Macaca fascicularis.

Neutropenia is a common consequence of radiation and chemotherapy in cancer patients. The resulting immunocompromised patients become highly susceptible to potentially life-threatening infections. Granulocyte colony-stimulating factor (G-CSF) is known to stimulate neutrophil production and is widely used as a treatment of chemotherapy-induced neutropenia. A small-molecule G-CSF secretagogue without a requirement for refrigerated supply chain would offer a more convenient and cost-effective treatment of chemotherapy-induced neutropenia. Bacterial lipopeptides activate innate immune responses through Toll-like receptor 2 (TLR2) and induce the release of cytokines, including G-CSF, from macrophages, monocytes, and endothelial. Pam2CSK4 is a synthetic lipopeptide that effectively mimics bacterial lipoproteins known to activate TLR2 receptor signaling through the TLR2/6 heterodimer. Substrate-based drug design led to the discovery of GSK3277329, which stimulated the release of G-CSF in activated THP-1 cells, peripheral blood mononuclear cells, and human umbilical vein endothelial cells. When administered subcutaneously to cynomolgus monkeys (Macaca fascicularis), GSK3277329 caused systemic elevation of G-CSF and interleukin-6 (IL-6), but not IL-1ß or tumor necrosis factor α, indicating a selective cytokine-stimulation profile. Repeat daily injections of GSK3277329 in healthy monkeys also raised circulating neutrophils above the normal range over a 1-week treatment period. More importantly, repeated daily injections of GSK3277329 over a 2-week period restored neutrophil loss in monkeys given chemotherapy treatment (cyclophosphamide, Cytoxan). These data demonstrate preclinical in vivo proof of concept that TLR2 agonism can drive both G-CSF induction and subsequent neutrophil elevation in the cynomolgus monkey and could be a therapeutic strategy for the treatment of chemotherapy-induced neutropenia.

Discovery of 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H- pyran-4-yl)benzamide (GW788388): a potent, selective, and orally active transforming growth factor-beta type I receptor inhibitor.

Inhibitors of transforming growth factor beta (TGF-beta) type I receptor (ALK5) offer a novel approach for the treatment of fibrotic diseases such as renal, hepatic, and pulmonary fibrosis. The optimization of a novel phenylpyridine pyrazole series (1a) led to the identification of potent, selective, and orally active ALK5 inhibitors. The cellular potency and pharmacokinetics profiles of these derivatives were improved and several compounds presented antifibrotic activity when orally administered to rats in an acute liver model of dimethylnitrosamine- (DMN-) induced expression of collagen IA1 mRNA, a major gene contributing to excessive extra cellular matrix deposit. One of the most potent ALK5 inhibitors identified in this chemical series, compound 13d (GW788388), reduced the expression of collagen IA1 by 80% at a dose of 1 mg/kg twice a day (b.i.d.). This compound significantly reduced the expression of collagen IA1 mRNA when administered orally at 10 mg/kg once a day (u.i.d.) in a model of puromycin aminonucleoside-induced renal fibrosis.

ALK5 inhibition in renal disease.

Recent advances have identified novel small molecule inhibitors of the transforming growth factor-beta (TGF-beta) type I receptor kinase as a potential therapy in organ remodeling diseases, such as chronic renal disease. Because TGF-beta is central to the progression of fibrosis, selective inhibition of this signaling pathway could provide a novel treatment in many fibrotic diseases. The rationale for inhibition of TGF-beta signaling in renal disease includes prevention of fibrosis, tubular dedifferentiation and vascular effects.

SB-431542, a small molecule transforming growth factor-beta-receptor antagonist, inhibits human glioma cell line proliferation and motility.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that promotes malignant glioma invasion, angiogenesis, and immunosuppression. Antisense oligonucleotide suppression of TGF-beta(2) ligand expression has shown promise in preclinical and clinical studies but at least two ligands mediate the effects of TGF-beta in gliomas. Therefore, we examined the effects of SB-431542, a novel, small molecule inhibitor of the type I TGF-beta receptor, on a panel of human malignant glioma cell lines. SB-431542 blocked the phosphorylation and nuclear translocation of the SMADs, intracellular mediators of TGF-beta signaling, with decreased TGF-beta-mediated transcription. Furthermore, SB-431542 inhibited the expression of two critical effectors of TGF-beta-vascular endothelial growth factor and plasminogen activator inhibitor-1. SB-431542 treatment of glioma cultures inhibited proliferation, TGF-beta-mediated morphologic changes, and cellular motility. Together, our results suggest that small molecule inhibitors of TGF-beta receptors may offer a novel therapy for malignant gliomas by reducing cell proliferation, angiogenesis, and motility.

Identification of novel inhibitors of the transforming growth factor beta1 (TGF-beta1) type 1 receptor (ALK5).

Screening of our internal compound collection for inhibitors of the transforming growth factor beta1 (TGF-beta1) type I receptor (ALK5) identified several hits. Optimization of the dihydropyrroloimidazole hit 2 by introduction of a 2-pyridine and 3,4-methylenedioxyphenyl group gave 7, a selective ALK5 inhibitor. With this information, optimization of the triarylimidazole hit 8 gave the selective inhibitor 14, which inhibits TGF-beta1-induced fibronectin mRNA formation while displaying no measurable cytotoxicity in the 48 h XTT assay.